Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/04—Ortho-condensed systems

Definitions

• the present disclosure belongs to the field of biomedicine, specifically related to a salt and crystal form of nitrogen-containing heterocyclic derivative, a preparation method therefor and an application thereof.
• Rat sarcomas encoded by proto-oncogenes HRAS, NRAS, and KRAS, is calssified as 4 proteins, HRAS, NRAS, KRAS4A and KRAS4B, and is a GTP (guanosine triphosphate) binding protein.
• RAS is located on the inner surface of a cell membrane, upstream of which is receptor tyrosine kinase (RTK), after activation, RAS regulates downstream PI3K, RAF and other signaling pathways, thereby regulating cell growth, survival, migration, differentiation, and other functions.
• RTK receptor tyrosine kinase
• RAS has two main states in the body: an inactivated state combined with GDP (guanosine diphosphate) and an activated state combined with GTP. Its activity is regulated by two proteins, guanine nucleotide exchange factor (GEF) promotes the release of GDP from the RAS protein, allowing GTP to bind to activate RAS; GTPase activating protein (GAP) activates the GTPase activity of RAS protein, hydrolyzes the GTP bound to RAS protein into GDP, and inactivates the RAS. Under normal circumstances, the RAS protein is in a non-activated state, the conformation changes after mutation, and the RAS is in a continuously activated state, and downstream signaling pathways are also continuously activated, leading to the occurrence of various cancers.
• GEF guanine nucleotide exchange factor
• GAP GTPase activating protein
• RAS is the oncogene with the highest mutation rate, accounting for an average of 25% of human cancers.
• the most common oncegenic mutation in the RAS family is KRAS (85%), while NRAS (12%) and HRAS (3%) are relatively rare.
• KRAS mutations mainly occur in a series of cancers such as pancreatic cancer (95%), colorectal cancer (52%) and lung cancer (31%), etc.
• the most common mutation mode of KRAS is point mutation, which mostly occurs in G12, G13 in p-loop (aa 10 to 17) and Q61 in Switch II region (aa 59 to 76), where G12 mutation is the most common (83%).
• KRAS G12C is one of the most common mutations in non-small cell lung cancer (NSCLC) and colorectal cancer.
• KRAS inhibitors are mainly due to two factors: first, the structure of RAS protein is smooth, and small molecules are difficult to bind to the protein surface; secondly, the affinity of RAS GTPase for GTP is as high as picomolar (pM) level, and the level of endogenous GTP is high, small molecule drugs are difficult to block the combination of the two.
• pM picomolar
• KRAS G12C inhibitors are expected to be the first drug directly targeting KRAS.
• KRAS G12C inhibitors have entered the clinical research stage, such as AMG 510 developed by Amgen, ARS-3248 developed by Wellspring Biosciences and MTRX849 developed by Mirati, all of which are currently in the clinical Phase I research stage, but none of them have been developed and marketed as KRAS G12C inhibitors yet.
• KRAS G12C There is no specific target drug for KRAS G12C, and there is a large clinical demand.
• the KRAS G12C inhibitors with higher selectivity, better activity and better safety have the potential to treat a variety of cancers, and have broad market prospects.
• the object of the present disclosure is to provide an acid salt of a compound represented by general formula (I): wherein:
• R a is each independently selected from hydrogen, deuterium, halogen, amino, hydroxyl, sulfhydryl, cyano, nitro, C 1-6 alkyl, C 1-6 deuterated alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, -SR aa , -C(O)R aa , -NR aa R bb , C 1-6 haloalkoxy or C 1-6 hydroxyalkyl;
• the compound is further shown in general formula (II-A) or (II-B):
• the acid salt of the compound wherein the compound is selected from: or
• the compound is selected from: P-4-((2 S ,5 R )-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one P-4-(( S )-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one 4-(( S )-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-3-chloro-2-fluorophenyl)-6-chloro
• Acid in the acid salt is selected from hydroxyethyl sulfonic acid, sulfuric acid, 1,5-naphthalene disulfonic acid, methanesulfonic acid, hydrobromic acid, phosphoric acid, benzenesulfonic acid, oxalic acid, maleate acid, adipic acid, hydrochloric acid, citric acid, malonic acid, L-malic acid, pamoic acid, p -toluenesulfonic acid or fumaric acid, preferably hydroxyethyl sulfonic acid or sulfuric acid.
• the number of acid is 0.2-3; preferably 0.2, 0.5, 1, 1.5, 2, 2.5 or 3; more preferably 0.5, 1, 2 or 3.
• the acid salt is a hydrate or an anhydrate, and when the acid salt is the hydrate, the number of water is 0.2-3; preferably 0.2, 0.5, 1, 1.5, 2, 2.5 or 3; more preferably 0.5, 1, 2 or 3.
• an acid salt of compound P-4-((2 S ,5 R )-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one wherein the acid in the acid salt is selected from hydroxyethyl sulfonic acid, sulfuric acid, 1,5-naphthalene disulfonic acid, methylsulfonic acid, hydrobromic acid, phosphoric acid, benzenesulfonic acid, oxalic acid, maleate acid, adipic acid, hydrochloric acid, citric acid, malonic acid, L-malic acid, pamoic acid, p -toluenesulfonic acid or fumaric
• the acid salt is in a crystal form; preferably a crystal form of the acid salt of compound P-4-((2 S ,5 R )-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1 H )-one;
• the crystal form of the acid salt of compound P-4-((2 S ,5 R )-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one is provided.
• the acid salt of compound P-4-((2 S ,5 R )-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one is in a crystal form; preferably the crystal form of hydroxyethyl sulfonate, the crystal form of sulfate, the crystal form of 1,5-naphthalene disulfonate, the crystal form of methanesulfonate, the crystal form of hydrobromate, the crystal form of phosphate, the crystal form of benzenesulfonate, the crystal form of oxalate, the crystal form of maleate, the crystal form of adipate, the crystal form of hydrochloride,
• the acid salt is in a crystal form; wherein the number of acid is 0.2-3; preferably 0.2, 0.5, 1, 1.5, 2, 2.5 or 3; more preferably 0.5, 1, 2 or 3.
• crystal forms I-III of hydroxyethyl sulfonate and crystal forms I-IV of sulfate of compound P-4-((2 S ,5 R )-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one are provided:
• crystal forms I-III of hydroxyethyl sulfonate and crystal forms I-IV of sulfate of compound P-4-((2 S ,5 R )-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one are provided:
• crystal forms I-III of hydroxyethyl sulfonate and crystal forms I-IV of sulfate of compound P-4-((2 S ,5 R )-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one are provided:
• the crystal form I of hydroxyethyl sulfonate of compound P-4-((2 S ,5 R )-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one shown in embodiment 13-1 described in the present disclosure has an X-ray powder diffraction pattern basically as shown in FIG. 1 ; a DSC pattern basically as shown in FIG. 2 ; and a TGA pattern basically as shown in FIG. 3 .
• the crystal form II of hydroxyethyl sulfonate of compound P-4-((2 S ,5 R )-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one shown in embodiment 13-1 described in the present disclosure has an X-ray powder diffraction pattern basically as shown in FIG. 4 ; a DSC pattern basically as shown in FIG. 5 ; and a TGA pattern basically as shown in FIG. 6 .
• the crystal form III of hydroxyethyl sulfonate of compound P-4-((2 S ,5 R )-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one shown in embodiment 13-1 described in the present disclosure has an X-ray powder diffraction pattern basically as shown in FIG. 7 ; a DSC pattern basically as shown in FIG. 8 ; and a TGA pattern basically as shown in FIG. 9 .
• the crystal form I of sulfate of compound P-4-((2 S ,5 R )-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one shown in embodiment 13-1 described in the present disclosure has an X-ray powder diffraction pattern basically as shown in FIG. 10 .
• the crystal form II of sulfate of compound P-4-((2 S ,5 R )-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one shown in embodiment 13-1 described in the present disclosure has an X-ray powder diffraction pattern basically as shown in FIG. 11 .
• the crystal form III of sulfate of compound P-4-((2 S ,5 R )-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one shown in embodiment 13-1 described in the present disclosure has an X-ray powder diffraction pattern basically as shown in FIG. 12 .
• positions of diffraction peaks with relative peak intensity of top ten in the X-ray powder diffraction pattern of the crystal form I of hydroxyethyl sulfonate and diffraction peaks at the corresponding positions in FIG. 1 have a 2 ⁇ error of ⁇ 0.2° to ⁇ 0.5°, preferably ⁇ 0.2° to ⁇ 0.3°, most preferably ⁇ 0.2°;
• the crystal form of the acid salt is a hydrate or an anhydrate
• the number of water is 0.2 to 3, preferably 0.2, 0.5, 1, 1.5, 2, 2.5 or 3, more preferably 0.5, 1, 2 or 3; further, the water in the hydrate is pipeline water, or crystal water, or a combination of both.
• a method for preparing an acid salt comprises the following steps:
• a method for preparing the acid salt of the compound and a crystal form thereof comprises the following steps:
• a method for preparing a crystal form of the acid salt of the compound comprises the following steps:
• a method for preparing the acid salt of the compound or the crystal form thereof comprises the following steps:
• the reaction solvent used in step 1) is an organic solvent, preferably at least one of ethanol, propanol, isopropanol, 2-methyltetrahydrofuran, n-heptane, methyl tert -butyl ether, toluene, isopropyl acetate, tert -butanol, n -butanol, tetrahydrofuran, acetone, 2-butanone, ethyl acetate or 1,4-dioxane;
• the acid in step 2) is selected from hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, phosphoric acid, 2,5-dihydroxybenzoic acid, 1-hydroxy-2-naphthoic acid, acetic acid, ethanesulfonic acid, dichloroacetic acid, trichloroacetic acid, acetohydroxamic acid, adipic acid,
• the organic solvent in step 2) is selected from one or more of alcohol, ether, ketone or ester solvents, preferably at least one of ethanol, propanol, isopropanol, 2-methyltetrahydrofuran, n -heptane, methyl- tert -butyl ether, toluene, isopropyl acetate, tert- butanol, n-butanol, tetrahydrofuran, acetone, 2-butanone, ethyl acetate or 1,4-dioxane;
• the solvent in step 3) is selected from one or more of alcohol, ether, ketone or ester solvents, preferably at least one of ethanol, propanol, isopropanol, 2-methyltetrahydrofuran, n- heptane, methyl tert -butyl ether, toluene, isopropyl acetate, tert -butan
• the present disclosure also provides a preferred embodiment, and relates to a pharmaceutical composition
• a pharmaceutical composition comprising a therapeutically effective amount of the acid salt of the compound represented by general formula (I) or the crystal form thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.
• the present disclosure further relates to a use of any one of the acid salts of the compound represented by general formula (I) or the crystal forms thereof, or the pharmaceutical composition in the manufacture of a medicament of a KRAS inhibitor; preferably a use in the manufacture of a medicament of a KRAS G12C mutation inhibitor.
• the pharmaceutically acceptable salt of the compound and the crystal form thereof, or the composition of the present disclosure is used for treating Noonan syndrome, leopard syndrome, leukemia, neuroblastoma, melanoma, esophagus cancer, head and neck tumor, breast cancer, lung cancer and colon cancer; preferably non-small cell lung cancer, colon cancer, esophagus cancer, and head and neck tumor.
• alkyl refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably alkyl containing 1 to 8 carbon atoms, more preferably alkyl containing 1 to 6 carbon atoms, most preferably alkyl containing 1 to 3 carbon atoms.
• Non-limiting examples include methyl, ethyl, n -propyl, isopropyl, n -butyl, isobutyl, tert -butyl, sec -butyl, n -pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n -hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n -heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl,
• lower alkyl containing 1 to 6 carbon atoms non-limiting examples include methyl, ethyl, n -propyl, isopropyl, n -butyl, isobutyl, tert -butyl, sec-butyl, n -pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n -hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc.
• the alkyl may be substituted or unsubstituted, when substituted, the substituents may be substituted at any available attachment point, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxyl, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylate, preferably alkyl substituted by methyl, ethyl, isopropyl, tert-butyl, haloalkyl, deuterated alkyl, alkoxy-substituted alkyl and hydroxyl-substituted alkyl.
• alkylene refers to that one hydrogen atom of alkyl is further substituted, for example: "methylene” refers to -CH 2 -, "ethylene” refers to -(CH 2 ) 2 -, and “propylene” refers to -(CH 2 ) 3 -, “butylene” refers to -(CH 2 ) 4 -, etc.
• alkenyl refers to alkyl as defined above containing at least two carbon atoms and at least one carbon-carbon double bond, such as vinyl, 1-propenyl, 2-propenyl, 1-, 2-, or 3 -butenyl etc.
• the alkenyl may be substituted or unsubstituted, when substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.
• cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 6 carbon atoms.
• Non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctanyl, etc.; polycylic cycloalkyl includes spiro, fused and bridged cycloalkyl, preferably cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl and cycloheptyl.
• spirocycloalkyl refers to polycyclyl that shares one carbon atom (called a spiro atom) between 5- to 20-membered monocyclic rings, which may contain one or more double bonds, but none of the rings has a fully conjugated ⁇ -electron system. Preferably 6-14-membered, more preferably 7-10-membered. According to the number of shared spiro atoms between the rings, the spirocycloalkyl is classified into monospirocycloalkyl, bispirocycloalkyl or polyspirocycloalkyl, preferably monospirocycloalkyl and bispirocycloalkyl.
• spirocycloalkyl More preferably, 3-membered/6-membered, 3-membered/5-membered, 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered, or 5-membered/6-membered monospirocycloalkyl.
• spirocycloalkyl include: etc.; also include spirocycloalkyl in which monospirocycloalkyl and heterocycloalkyl share a spiro atom, and non-limiting examples include: etc.
• fused cycloalkyl refers to a 5-20-membered all-carbon polycyclic group in which each ring in the system shares an adjacent pair of carbon atoms with other rings in the system, wherein one or more of the rings may comprise one or multiple double bonds, but none of the rings has a fully conjugated ⁇ -electron system.
• 6-14-membered more preferably 7-10-membered.
• it can be classified into bicyclic, tricyclic, tetracyclic or polycyclic fused cycloalkyl, preferably bicyclic or tricyclic, and more preferably 5-membered/5-membered or 5-membered/6-membered bicyclic alkyl.
• fused cycloalkyls include: etc.
• bridged cycloalkyl refers to 5 to 20-membered all-carbon polycyclic group, in which any two rings share two carbon atoms that are not directly connected, it may contain one or more double bonds, but none of the rings has a fully conjugated ⁇ -electron system. Preferably 6-14-membered, more preferably 7-10-membered. According to the number of constituent rings, it can be classified into bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl, preferably bicyclic, tricyclic, or tetracyclic, and more preferably bicyclic or tricyclic.
• bridged cycloalkyl include:
• the cycloalkyl ring may be fused to an aryl, heteroaryl or heterocycloalkyl ring, wherein the ring connected to the parent structure is cycloalkyl, non-limiting examples include indanyl, tetrahydronaphthyl, benzocycloheptanyl, etc.
• the cycloalkyl may be substituted or unsubstituted, when substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboylate.
• heterocyclyl refers to saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, wherein one or more of the ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) m (wherein m is an integer of 0 to 2), but not including the ring part of -O-O-, -O-S- or -S-S-, and the remaining ring atoms are carbon.
• It preferably contains 3 to 12 ring atoms, wherein 1 to 4 ring atoms are heteroatoms; more preferably contains 3 to 8 ring atoms; most preferably contains 3 to 8 ring atoms; further preferably 3-8-membered heterocyclyl containing 1 to 3 nitrogen atoms, optionally substituted by 1 to 2 oxygen atoms, sulfur atoms or oxo, including nitrogen-containing monocyclic heterocyclyl, nitrogen-containing spiro heterocyclyl or nitrogen-containing fused heterocyclyl.
• Non-limiting examples of monocyclic heterocyclyl include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, azepyl, 1,4-diazepanyl, pyranyl, etc., preferably pyrrolidinyl, morpholinyl, piperidinyl, azepanyl, 1,4-diazepanyl and piperazinyl.
• Polycyclic heterocyclyl include spiro-, fused- and bridged heterocyclyl; the spiro-, fused- and bridged heterocyclyl are optionally connected to other groups through a single bond, or to connect to other cycloalkyl, heterocyclyl, aryl and heteroaryl through any two or more of ring atoms.
• spiroheterocyclyl refers to polycyclic heterocyclyl sharing one atom (called a spiro atom) between 5-20-membered monocyclic ring, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) m (wherein m is an integer of 0 to 2) heteroatoms, and the remaining ring atoms are carbon. It may contain one or more double bonds, but none of the rings has fully conjugated ⁇ -electron system. Preferably 6-14-membered, more preferably 7-10-membered.
• the spiro heterocyclyl is classified into monospiroheterocyclyl, dispiroheterocyclyl or polyspiroheterocyclyl, preferably monospiroheterocyclyl and dispiroheterocyclyl. More preferably, 3-membered/5-membered, 3-membered/6-membered, 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered, or 5-membered/6-membered monospiroheterocyclyl.
• Non-limiting examples of spiroheterocyclyl include: etc.
• fused heterocyclyl refers to a 5-20-membered polycyclic heterocylic group in which each ring in the system shares an adjacent pair of atoms with other rings in the system, one or more of the rings may comprise one or multiple double bonds, but none of the rings has a fully conjugated ⁇ -electron system, wherein one or more of the ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) m (wherein m is an integer of 0 to 2), the rest of the ring atoms are carbon.
• m is an integer of 0 to 2
• the number of constituent rings it can be classified into bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclyl, preferably bicyclic or tricyclic, and more preferably 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocylyl.
• fused heterocylyl include: etc.
• bridged heterocyclyl refers to polycyclic heterocylic group in which any two rings share two atoms that are not directly connected, it may contain one or multiple double bonds, but none of the rings has a fully conjugated ⁇ -electron system, wherein one or more of the ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) m (wherein m is an integer of 0 to 2), the rest of the ring atoms are carbon.
• nitrogen, oxygen or S(O) m wherein m is an integer of 0 to 2
• m is an integer of 0 to 2
• 6-14-membered Preferably 6-14-membered, more preferably 7-10-membered.
• the number of constituent rings it can be classified into bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclyl, preferably bicyclic, tricyclic, or tetracyclic, and more preferably bicyclic or tricyclic.
• bridged heterocylyl include: etc.
• the heterocyclic ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring connected to the parent structure is heterocyclyl, and non-limiting examples of heterocyclyl include: etc.
• the heterocyclyl may be substituted or unsubstituted, when substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboylate.
• aryl refers to a 6-14-membered all-carbon monocyclic or fused polycyclic (that is, rings sharing adjacent pairs of carbon atoms) with conjugated ⁇ -electron system, preferably 6-12-membered, such as phenyl and naphthyl. More preferably phenyl.
• the aryl ring may be fused on a heteroaryl, heterocyclyl or cycloalkyl ring, including benzo 5-10-membered heteroaryl, benzo 3-8-membered cycloalkyl and benzo 3-8-membered heteroalkyl, preferably benzo 5-6-membered heteroaryl, benzo 3-6-membered cycloalkyl and benzo 3-6-membered heteroalkyl, wherein the heterocyclyl is heterocyclyl containing 1 to 3 nitrogen atoms, oxygen atoms and sulfur atoms; or a 3-membered nitrogen-containing fused ring containing a benzene ring.
• ring connected to the parent structure is an aryl ring, and non-limiting examples of aryl include: etc.
• the aryl may be substituted or unsubstituted, when substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalky, heterocycloalky, aryl, heteroaryl, cycloalkoxyl, heterocycloalkoxyl, cycloalkylthio, heterocycloalkylthio, carboxyl or carboylate.
• groups are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalky, heterocycloalky, aryl, heteroaryl, cycloalkoxyl, heterocycloalkoxyl,
• heteroaryl refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen.
• the heteroaryl is preferably 5-12-membered, more preferably 5- or 6-membered, such as imidazole, furanyl, thiophenyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, pyrazinyl, etc., preferably triazolyl, thiophenyl, imidazolyl, pyrazolyl, oxazolyl, pyrimidinyl or thiazolyl; more preferably pyrazolyl, pyrrolyl and oxazolyl.
• the heteroaryl ring may be fused to an aryl, heteroaryl or cycloal
• the heteroaryl may be optionally substituted or unsubstituted, when substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalky, heterocycloalky, aryl, heteroaryl, cycloalkoxyl, heterocycloalkoxyl, cycloalkylthio, heterocycloalkylthio, carboxyl or carboylate.
• groups are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalky, heterocycloalky, aryl, heteroaryl, cycloalkoxyl, heterocycloalkoxyl
• alkoxy refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein the definition of alkyl is as described above, preferably alkyl containing 1 to 8 carbon atoms, more preferably alkyl containing 1 to 6 carbon atoms, most preferably alkyl containing 1 to 3 carbon atoms.
• alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy.
• the alkoxy may be optionally substituted or unsubstituted, when substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboylate.
• groups are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy
• alkylthio refers to -S-(alkyl) and -S-(unsubstituted cycloalkyl), wherein the definition of alkyl is as described above.
• alkyl containing 1 to 8 carbon atoms Preferably alkyl containing 1 to 6 carbon atoms, most preferably alkyl containing 1 to 3 carbon atoms.
• alkylthio include: methylthio, ethylthio, propylthio, butylthio, cyclopropylthio, cyclobutylthio, cyclopentylthio, cyclohexylthio.
• the alkylthio may be optionally substituted or unsubstituted, when substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboylate.
• Alkylthio-alkyl refers to alkylthio attached to alkyl, wherein the alkyl and the alkylthio are as defined above.
• Alkylaminocarbonyl refers to (alkyl)-N-C(O)-, wherein the alkyl is as defined above.
• Haloalkyl refers to alkyl substituted by one or more halogens, wherein the alkyl is as defined above.
• Haloalkoxy refers to alkoxy substituted by one or more halogens, wherein the alkoxy is as defined above.
• Haloalkoxy refers to alkylthio substituted by one or more halogens, wherein the alkylthio is as defined above.
• Hydroalkyl refers to alkyl substituted by one or more hydroxyl, wherein the alkyl is as defined above.
• Alkenyl refers to chain alkenyl, also known as alkylene, preferably alkyl containing 2 to 8 carbon atoms, more preferably alkyl containing 2 to 6 carbon atoms, most preferably alkyl containing 2 to 3 carbon atoms.
• alkenyl may be further substituted with other related groups, such as: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalky, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.
• other related groups such as: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalky, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate
• alknyl may be further substituted by other related groups, for example: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboylate.
• alkenylcarbonyl refers to -C(O)-(alkenyl), wherein the alkenyl is as defined above.
• alkenylcarbonyl include: vinylcarbonyl, propenylcarbonyl, butenylcarbonyl.
• the alkenylcarbonyl may be optionally substituted or unsubstituted, when substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboylate.
• Hydrophill refers to an -OH group.
• Halogen refers to fluorine, chlorine, bromine or iodine.
• Amino refers to -NH 2 .
• Cyano refers to -CN.
• Niro refers to -NO 2 .
• Carbonyl refers to -C(O)-.
• Carboxyl refers to -C(O)OH.
• THF tetrahydrofuran
• EtOAc refers to ethyl acetate
• MeOH refers to methanol
• DIPEA diisopropylethylamine
• TFA trifluoroacetic acid
• MeCN refers to acetonitrile
• DMA refers to N,N -dimethylacetamide.
• Et 2 O refers to diethyl ether
• DIPEA N,N- diisopropylethylamine
• NBS N -bromosuccinimide
• NIS N -iodosuccinimide
• Cbz-Cl refers to benzyl chloroformate
• Pd 2 (dba) 3 refers to tris(dibenzylideneacetone)dipalladium.
• Dppf refers to 1,1'-bis(diphenylphosphino)ferrocene.
• HATU refers to 2-(7-azabenzotriazol-1-yl)- N,N,N ', N '-tetramethyluronium hexafluorophosphate.
• KHMDS refers to potassium hexamethyldisilazide
• LiHMDS refers to lithium bistrimethylsilylamide.
• MeLi refers to methyl lithium
• N-BuLi refers to n -butyl lithium.
• NaBH(OAc) 3 refers to sodium triacetoxyborohydride.
• X is selected from A, B, or C
• X is selected from A, B and C
• X is A, B or C
• X is A, B and C
• other terms all express the same meaning, which means that X can be any one or more of A, B, and C.
• the hydrogen atom described in the present disclosure may be replaced by its isotope deuterium, and any hydrogen atom in the compounds according to the embodiments of the present disclosure may also be replaced by a deuterium atom.
• heterocyclic group optionally substituted by alkyl refers to that alkyl may, but not necessarily, be present, and the description includes the case where the heterocyclic group is substituted by alkyl and the case where the heterocyclic group is not substituted by alkyl.
• Substituted refers to one or more hydrogen atoms in the group, preferably up to 5, more preferably 1 to 3 hydrogen atoms, independently substituted by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine possible or impossible substitutions (by experiment or theory) without too much effort. For example, amino or hydroxyl having free hydrogen may be unstable when combined with a carbon atom having an unsaturated (e.g., olefinic) bond.
• “Pharmaceutical composition” refers to a mixture containing one or more of the compounds described herein or the physiologically/pharmaceutically acceptable salt or prodrug thereof and other chemical components, and the other component is, for example, physiological/pharmaceutically acceptable carrier and excipient.
• the purpose of the pharmaceutical composition is to promote the administration to an organism, facilitate the absorption of an active ingredient and then exert the biological activity.
• “Pharmaceutically acceptable salt” refers to the salt of the compound of the present disclosure, which is safe and effective when used in mammals, and has due biological activity.
• NMR nuclear magnetic resonance
• LC-MS liquid chromatography-mass spectrometry
• Liquid chromatography-mass spectrometry LC-MS was determined with an Agilent 1200 Infinity Series mass spectrometer. HPLC determinations were performed using an Agilent 1200DAD high pressure liquid chromatograph (Sunfire C18 150 ⁇ 4. 6 mm column) and a Waters 2695-2996 high pressure liquid chromatograph (Gimini C18 150 ⁇ 4. 6 mm column).
• Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate was used as a thin-layer chromatography silica gel plate, the specification of TLC was 0.15 mm to 0.20 mm, and the specification of thin-layer chromatography separation and purification products was 0.4 mm to 0.5 mm.
• Yantai Huanghai silica gel 200 to 300 mesh silica gel was used as a carrier for column chromatography.
• the starting materials in the embodiments of the present disclosure are known and commercially available, or can be synthesized by using or following methods known in the art.
• 2,4-Dichloropyridin-3-amine (4.5 g, 27.78 mmol), 4,4,5,5-tretramethyl-2-(prop-1-en-2-yl)-1,3,2-dioxaborolane (5.13 g, 30.56 mmol), potassium carbonate (11.5 g, 83.34 mmol), Pd(PPh 3 ) 4 were added to dioxane (120 mL), and the reaction mixture was uniformly mixed and then stirred overnight in an oil bath at 100°C.
• Step 4 Preparation of 2,6-dichloro-5-fluoro- N -((2-isopropyl-4-(methylthio)pyridin-3-yl)carbamoyl)nicotinamide
• Step 5 Preparation of 7-chloro-6-fluoro-4-hydroxy-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one
• Step 6 Preparation of tert -butyl ( S )-4-(7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-carbonyl-1,2- d ]hydropyrido[2,3- d ]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate
• Step 7 Preparation of ( S )-4-(4-acryloyl-2-methylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one
• Step 8 Preparation of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one
• Step 1 Preparation of 4,7-dichloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1 H )-one
• N,N- Diisopropylethylamine (407 mg, 3.16 mmol) was added to a solution of 7-chloro-6-fluoro-4-hydroxy-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one (200 mg, 0.526 mmol) in acetonitrile (10 mL) at room temperature, then phosphorus oxychloride (242 mg, 1.58 mmol) was added thereto and the mixture was stirred at 80°C for 1 hour. The mixture was cooled to room temperature and directly used in the next reaction.
• Step 2 Preparation of tert -butyl (2 R ,5 S )-4-(7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-carbonyl-1,2-dihydropyrido[2,3- d ]pyrimidin-4-yl)-2,5-dimethylpiperazine-1-carboxylate
• N,N-Diisopropylethylamine (678 mg, 5.26 mmol) and tert -butyl (2 R ,5 S )-2,5-dimethylpiperazine-1-carboxylate (224 mg, 1.005 mmol) were added to the reaction mixture of the previous step and stirred for 1 hour at room temperature after the addition.
• Step 3 Preparation of 7-chloro-4-((2 S ,5 R )-2,5-dimethylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one trifluoroacetate
• Trifluoroacetic acid (1.2 mL) was added to a solution of tert -butyl (2 R ,5 S )-4-(7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-carbonyl-1,2-dihydropyrido[2,3- d ]pyrimidin-4-yl)-2,5-dimethylpiperazine-1-carboxylate (200 mg, 0.347 mmol) in dichloromethane (6 mL), and the mixture was stirred at room temperature for 1.5 hours after the addition.
• reaction mixture was concentrated at low temperature to obtain 7-chloro-4-((2 S ,5 R )-2,5-dimethylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one trifluoroacetate (200 mg) as a red oil, which was used rapidly in the next reaction.
• Step 4 Preparation of 4-((2 S ,5 R )-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1H)-one
• N,N -Diisopropylethylamine (447 mg, 3.47 mmol) was added to a solution of 7-chloro-4-((2 S ,5 R )-2,5-dimethylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1H)-one trifluoroacetate (200 mg, 0.347 mmol) in dichloromethane (15 mL), then acryloyl chloride (63 mg, 0.694 mmol) was added dropwise thereto at 0°C, and the mixture was stirred for 1 hour after the addition.
• Step 5 Preparation of 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one
• Step 1 Preparation of N -(4-chloro-3-fluorophenyl)-2,2,2-trifluoroacetamide
• N -(4-Chloro-3-fluorophenyl)-2,2,2-trifluoroacetamide (2.3 g, 9.5 mmol) was dissolved in THF (40 mL), the mixture was cooled to -78°C under nitrogen atmosphere, and n- BuLi (7.9 mL, 19.0 mmol, 2.4 M) was added dropwise thereto, then the mixture was stirred at -50°C for 50 minutes after the addition.
• reaction mixture was cooled to -78°C, triisopropyl borate (2.3 g, 9.5 mmol) (4.8 mL, 20.9 mmol) was added dropwise thereto, the mixture was stirred at the same temperature for 20 minutes after the addition, the dry ice bath was removed, and the mixture was stirred at room temperature for 2 hours. Then, the reaction mixture was cooled to 0°C, dilute hydrochloric acid (19 mL, 1M) was added dropwise thereto, the temperature was raised to 40°C, and the mixture was stirred for 1 hour. The mixture was then extracted three times with ethyl acetate (100 mL).
• Step 4 Preparation of 4-(( S )-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-6-fluorophenyl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one
• Embodiment 9-1 and embodiment 9-2 are identical to Embodiment 9-1 and embodiment 9-2.
• Embodiment 9 was resolved by SFC to obtain two axial chiral isomers, embodiment 9-1 and embodiment 9-2, SFC: chiral preparation conditions: Instrument SFC-80 (Thar, Waters) Column type IC 20*250 mm, 10 ⁇ m (Daicel) Column pressure 100 bar Mobile phase CO 2 / Methanol (0.2% Methanol Ammonia) Flow rate 80 g/min Detection wavelength UV 214 nm Column temperature 35°C
• Step 1 Preparation of 2,5,6-trichloro -N -(2-isopropyl-4-(methylthio)pyridin-3-yl)carbamoyl)nicotinamide
• Step 2 Preparation of 6,7-dichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2,4(1 H ,3 H )-dione
• Step 3 Preparation of 4,6,7-trichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one
• Step 4 Preparation of tert -butyl (2 R ,5 S )-4-(6,7-dichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-carbonyl-1,2-dihydropyrido[2,3- d ]pyrimidin-4-yl)-2,5-dimethylpiperazine-1-carboxylate
• Step 5 Preparation of 6,7-dichloro-4-((2 S ,5 R )-2,5-dimethylpiperazin-1-yl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one
• Step 6 Preparation of 4-((2 S ,5 R )-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6,7-dichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one
• Step 7 Preparation of 4-((2 S ,5 R )-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d ]pyrimidin-2(1 H )-one
• Embodiment 13 was resolved by SFC to obtain two axial chiral isomers, embodiment 13-1 and embodiment 13-2, SFC: chiral preparation conditions: Instrument SFC-150 (Thar, Waters) Column type IC 20*250 mm, 10 ⁇ m (Daicel) Column pressure 100 bar Mobile phase CO 2 / Methanol (0.2% Methanol Ammonia) Flow rate 120 g/min Detection wavelength UV 214 nm Column temperature 35°C
• Embodiment 14 was resolved by SFC to obtain two axial chiral isomers, embodiment 14-1 and embodiment 14-2, SFC: chiral preparation conditions: Instrument SFC-150 (Thar, Waters) Column type IC 20*250 mm, 10 ⁇ m (Daicel) Column pressure 100 bar Mobile phase CO 2 / Methanol (0.2% Methanol Flow rate 120 g/min Detection wavelength UV 214 nm Column temperature 35°C
• NCI-H358 or Mia PaCa-2 cells were cultured to the appropriate fusion level, the NCI-H358 or Mia PaCa-2 cells were collected, and the cells were adjusted to the appropriate cell concentration using a complete medium, and the cell suspension was spread in a 96-well plate, 90 ⁇ L per well, and placed in a 37°C, 5% CO 2 incubator overnight; and compound solutions of different concentrations were prepared using DMSO and culture medium; and a solvent control was set, the compound solution was added to a 96-well plate, 10 ⁇ L per well, at 37°C in a 5% CO 2 incubator for 72 hours; CellTiter-Glo solution was added thereto and the mixture was mixed well by shaking, incubated for 10 min in the dark, and read by BioTek Synergy H1 microplate reader.
• the luminescence signal values were used to calculate the inhibition rate, the concentration and the inhibition rate were fitted to a nonlinear regression curve using Graphpad Prism software, then the IC 50 value was obtained.
• the compounds of the embodiments of the present disclosure have a good inhibitory effect on the proliferation of NCI-H358 and Mia PaCa-2 cells.
• the degree of increase in protein melting temperature can be used to characterize the compound's ability to bind to KRAS G12C protein.
• the thermal shift method was used to test the degree of change in the melting temperature (Tm) of the KRAS G12C protein before and after the binding of the compound, in order to characterize the ability of the compound to improve the stability of the KRAS G12C protein.
• the specific experiment operation was as follows: A solution containing 20 ⁇ M HEPES (pH 7.5), 1 mM DTT, 5X SYPRO Orange and 150 mM NaCl was prepared as the experimental buffer, and a final concentration of 5.37 ⁇ M human KRAS G12C protein was added thereto.
• the reaction mixture was divided into 8 rows of PCR tubes, each 19.5 ⁇ L, and 0.5 ⁇ L of the test compound or DMSO were added respectively, so that the total reaction system was 20 ⁇ L, the final concentration of the compound was 10 ⁇ M, and 2.5% DMSO was set as the solvent control.
• the experimental data file of PCR instrument was imported into thermal shift software, and the melting temperature (Tm) of each treatment group was obtained, and the change value of melting temperature ( ⁇ Tm) was obtained by subtracting the Tm of DMSO solvent control group.
• the compounds of the present disclosure show the ability to increase the melting temperature of the protein as shown in Table 9 in the experiment of improving the binding stability of KRAS G12C protein.
• Table 9 Embodiment number Tm (°C) DMSO Tm (°C) ⁇ Tm (°C ) Embodiment 1 48.6 60.2 11.6 Embodiment 2 48.7 57.2 8.5 Embodiment 3 50.6 61.5 10.9 Embodiment 4 49.5 61.2 11.7 Embodiment 5 48.6 64.4 15.8 Embodiment 9 46.8 60.2 13.4 Embodiment 13 47.0 58.0 11.0
• Mia PaCa-2 cells were cultured to the appropriate fusion level, Mia PaCa-2 cells were collected, and the cell density was adjusted to 1 ⁇ 10 6 /mL using complete culture medium, the cell suspension was spread on a 96-well plate, 50 ⁇ L per well, and placed adherent to the wall in a 37°C, 5% CO 2 incubator overnight, compound solutions with different concentrations were prepared using DMSO and complete culture medium, a solvent control was set, the compound solution was added to a 96-well plate, 25 ⁇ L per well, and placed in a 37°C, 5% CO 2 incubator for 2 hours of continuous culture, the supernatant was discarded from the cell culture plate, 50 ⁇ L of lysis solution was added to each well, and lysing was performed for 30 minutes by shaking at room temperature, then the mixture was centrifuged at 1000 rpm for 1 minute, 15 ⁇ L of supernatant was transferred to 384 well plate, 5 ⁇ L of detection mixture (Eu-labeled anti-ERK 1/2
• the ratio of the signal values at 665 nm and 620 nm emission wavelength were calculated, and the ratio was used to calculate the inhibition rate, the concentration and the inhibition rate were fitted to a nonlinear regression curve using Graphpad Prism software, then the IC 50 value was obtained.
• mice mice were administered (plasma) after oral administration using Balb/c mice as test animals.
• HEC Hydroxyethyl cellulose
• the compounds of the embodiments were weighed and added into 4-mL glass bottles, respectively, 2.4 mL of the solution was added, and ultrasound was performed for 10 minutes to obtain a colorless clear solution with a concentration of 1 mg/mL.
• Balb/C mice males; PO, after overnight fasting, respectively, at a dose of 10 mg/kg, administered in a volume of 10 mL/kg.
• Blood samples were collected before administration and 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h and 8 h after administration, the blood was placed in EDTA-2K tube, centrifuged at 4°C 6000 rpm for 6 min to separate plasma, and stored at -80°C; food was consumed 4 hours after drug administration.
• Table 11 Pharmacokinetic parameters of the compounds in mice Embodiment number Tmax (hr) Cmax (ng/mL) AUC 0- ⁇ (ng/mL*hr) T 1/2 (hr) MRT (hr) Embodiment 2-1 0.25 1823 1373 0.6 0.7 Embodiment 13-1 0.25 264 347 1.0 1.5
• mice BALB/c nude mice were used as the test animals, and the human pancreatic cancer cell MiaPaca 2 xenograft (CDX) model was used for in vivo pharmacodynamic experiments to evaluate the antitumor effects of the test compounds.
• CDX human pancreatic cancer cell MiaPaca 2 xenograft
• MiaPaca 2 cells were removed from the cell bank, revived and added to DMEM medium (containing 10% FBS, 1% Glu, 1% P/S) and incubated in a CO 2 incubator (incubator temperature was 37°C, CO 2 concentration was 5%). After the cells were spread to 80-90% of the bottom of the culture flask, the cells were continued to be cultured in the CO 2 incubator.
• the process was repeated until the number of cells met the in vivo pharmacological inoculation requirement, and the cells in logarithmic growth period were collected and counted with an automatic cell counter, resuspended with PBS and Matrigel (volume ratio 1: 1) according to the count results, made into a cell suspension (the density was 8 ⁇ 10 7 /mL), and placed in an ice box for use.
• mice BALB/c nude mice, female, 6-8 weeks old, weighing about 18-22 g.
• the mice were kept in an environment free of special pathogens and in a single ventilated cage with 5 mice in each cage. All cages, bedding and water were sterilized before use, and all animals had free access to standard certified commercial laboratory diets.
• Nude mice were labeled with disposable universal ear tags for mice and rats before the start of the experiment, and the skin of the inoculation site was disinfected with 75% medical alcohol before inoculation, 0.1 mL (containing 8* 10 6 cells) of MiaPaca 2 tumor cells were inoculated subcutaneously on the right back of each mouse. When the tumor volume reached 100- 200 mm 3 , the group administration was started.
• the tested compounds were administered daily by oral intragastric administration, dosage/frequency (6 mg/kg QD ⁇ 3w), and the efficacy of each group at the end of the experiment was shown in Table 5.
• the anti-tumor efficacy was determined by dividing the average tumor increased volume of the compound-treated animals by the average tumor increased volume of the untreated animals.
• Table 12 Pharmacodynamic parameters of the compounds in transplanted tumor mice Grouping Tumor volume (mm 3 , Mean ⁇ SD) ⁇ T / ⁇ C (%) TGI (%) Day 0 Day 21 Day 21 Day 21 Vehicle QD ⁇ 3w 178 ⁇ 30 868 ⁇ 234 - - Embodiment 2-1 177 ⁇ 38 67 ⁇ 34 -62.36 162.36 Embodiment 9-1 178 ⁇ 40 72 ⁇ 18 -59.56 159.56 Embodiment 13-1 178 ⁇ 34 41 ⁇ 19 -76.76 176.76
• mice BALB/c nude mice, 6-8 weeks old, female, purchased from Shanghai Xipuer-Bikai Experimental Animal Co., Ltd.
• TGI (%) [(1-(mean tumor volume at the end of the administration in a treatment group - mean tumor volume at the start of administration in the treatment group))/(mean tumor volume at the end of treatment in the solvent control group - mean tumor volume at the start of treatment in the solvent control group)] ⁇ 100%.
• TGI (%) [1-(mean tumor volume at the end of dosing in a treatment group - mean tumor volume at the beginning of dosing in the treatment group)/mean tumor volume at the beginning of dosing in the treatment group] ⁇ 100%.
• Table 13 Pharmacodynamic parameters of the compounds in transplanted tumor mice Grouping Tumor volume (mm 3 , Mean ⁇ SD) ⁇ T / ⁇ C (%) TGI (%) Day 0 Day 15 Day 15 Day 15 Vehicle QD ⁇ 3w 202 ⁇ 58 400 ⁇ 111 - - Embodiment 13-1 10 mpk 203 ⁇ 74 267 ⁇ 155 32.59 67.41 AMG-510 10 mpk 202 ⁇ 72 324 ⁇ 204 61.98 38.02
• CHO-hERG cells were cultured in a 175 cm 2 flask, when the cell density reached 60-80%, the culture medium was removed, the cells were washed with 7 mL PBS, and then digested with 3 mL Detachin.
• Table 14 Composition of intracellular fluid and extracellular fluid Reagent Extracellular fluid (mM) Intracellular fluid (mM) CaCl 2 2 5.374 MgCl 2 1 1.75 KCI 4 120 NaCl 145 - Glucose 10 - HEPES 10 10 EGTA - 5 Na-ATP - 4 pH 7.40 (adjusted with NaOH), Osmolarity ⁇ 305 mOsm 7.25 (adjusted with KOH), Osmolarity-290 mOsm
• the highest test concentration was 40 ⁇ M, in a total of 6 concentrations of 40, 13.33, 4.44, 1.48, 0.49 and 0.16 ⁇ M respectively.
• the experimental data were analyzed by XLFit software.
• the inhibition of drugs on the cardiac hERG potassium channel was the main cause of QT prolonged syndrome caused by drugs. It can be seen from the experimental results that the embodiment compound of the present disclosure had no obvious inhibitory effect on the cardiac hERG potassium ion channel, and can avoid the toxic and side effects to the heart at a high dose.
• the purpose of this experiment was to examine the stability of the compounds of the embodiments in mouse, rat, dog and human plasma.
• Animal or human whole blood was collected, then the blood was put into a test tube containing anticoagulant, centrifuged at 3500 rpm for 10 min, and the upper layer of pale yellow plasma was collected.
• the compound was weighed, the stock solution was prepared with DMSO and the working solution was prepared with 100 mM phosphate buffer.
• lovastatin 4.05 mg was weighed and diluted to 10 mM stock solution with 1 mL of DMSO; 10 ⁇ L of 10 mM stock solution was pipetted into 1 mL of 100 mM phosphate buffer to a final concentration of 100 ⁇ M.
• NADPH reduced nicotinamide adenine dinucleotide phosphate
• 0.25 mg/mL microsome 4 mL of 100 mM phosphate buffer was added to 50 ⁇ L of 20 mg/mL microsome and mixed well.
• the embodiment compound to be tested was weighed and diluted to 10 mM with DMSO and then diluted to 100 ⁇ M with 100 mM phosphate buffer.
• Table 17 Single point inhibition results of CYP enzyme of compounds of the embodiments No. IC 50 ( ⁇ M) 1A2 2C9 2C19 2D6 3A4-M 3A4-T Control 0.064 0.459 0.293 0.099 0.089 0.117 Embodiment 2-1 >100 84.6 >100 >100 19.0 >100 Embodiment 9-1 48.9 59.3 43.2 44.5 4.6 18.0 Embodiment 13-1 66.7 58.8 28.8 21.2 4.7 9.1 Note: Strong inhibition: IC 50 ⁇ 1 ⁇ M; moderate inhibition: 1 ⁇ M ⁇ IC 50 ⁇ 10 ⁇ M; weak inhibition: IC 50 > 10 ⁇ M
• the purpose of this experimental method was to detect the plasma protein binding of the compounds of the embodiments in plasma.
• Liquid-phase mass spectrometer centrifuge, vortexer, pipette, continuous liquid dispenser, 96-well plate, tissue homogenizer (for tissue sample analysis), 50% methanol aqueous solution, acetonitrile solution with internal standard, blank matrix (plasma, urine or tissue homogenate, etc.)
• the embodiment compound was prepared into a 1 mM solution A with DMSO.
• Solution A was added to the plasma solution and prepared into a 5 ⁇ M solution B.
• Table 18 Plasma protein binding rate of compounds of the embodiments No. Human Rat Mouse Dog Embodiment 2-1 98.0 90.5 88.4 82.6 Embodiment 9-1 99.8 94.9 90.1 98.7 Embodiment 13-1 99.7 97.9 93.9 98.7 Embodiment 14-1 96.8 95.4 96.3 92.5
• mice The pharmacokinetic behavior of the compound of the embodiment 13-1 and AMG-510 compound, administered orally at a dose of 6 mg/kg, in mice (plasma, tumor tissue and intestine) was studied using MiaPaca 2 tumor-bearing mice as test animals.
• Embodiment 13-1 of the present disclosure AMG-510 compound, self-made.
• Embodiment compound 13-1 and compound AMG-510 were weighed and dissolved in the solution, the mixture was shaken well, and ultrasound was performed for 15 minutes to obtain a uniform suspension with a concentration of 0.6 mg/mL.
• MiaPaca 2 tumor-bearing mice were administered at a dose of 6 mg/kg in a volume of 10 mL/kg, respectively, based on body weight p.o. after fasting (animals were not administered at point 0 h).
• mice were sacrificed with CO 2 , 0.5 mL blood was collected from the heart and placed in EDTA-2K tube, centrifuged at 4°C 6000 rpm for 6 min to separate plasma, and stored at -80°C; after the tumor tissues were weighing, placed in a 2 mL centrifuge tube and stored at-80°C.
• the duodenum, ileum and colon tissues were cut with scissors, the contents were removed and cleaned twice with PBS, after absorbing water with absorbent paper, they were weighed, placed in a 2 mL centrifuge tube and stored at -80°C.
• the ratio of exposure of the compound of the embodiment of the present disclosure in the tumor of the mouse to the exposure in the blood was higher than that of AMG-510, with longer T 1/2 and MRT.
• XRPD X-ray powder diffraction
• BRUKER D8 ADVANCE Diffracted ray CuK (40 kV, 40 mA) Scan rate 10°/min (2 ⁇ value) Scan range 4° to 40° (2 ⁇ value)
• DSC Differential scanning calorimetry
• Acid Solvent Phenomenon after formic acid Result 1 1.0 M sulfuric acid (methanol solution) ethanol change from dissolved clarification to suspension salt formation 2 ethyl acetate suspension salt formation 3 isopropanol precipitation after dissolved clarification salt formation 4 1.0 M hydroxyethyl sulfonic acid (methanol solution) n-butanol change from dissolved clarification to suspension salt formation 5 ethanol precipitation after salt formation dissolved clarification 6 acetone precipitation after dissolved clarification salt formation 7 2-butanone precipitation after dissolved clarification salt formation 8 ethyl acetate precipitation after dissolved clarification salt formation 9 1,4-dioxane precipitation after dissolved clarification salt formation 10 n-butanol precipitation after dissolved clarification salt formation 11 isopropanol precipitation after dissolved clarification salt formation 12 1.0 M hydroxyethyl sulfonic acid (methanol solution) tetrahydrofuran change from dissolved clarification salt formation to suspension 13 0.125 M 1,5-naphthalene disul
• a good solvent was selected, the acid was weighed, the good solvent was added thereto to prepare a stock solution containing the compound in the concentration of 100 mg/mL.
• An anti-solvent was added thereto, 100 mg of compound was weighed respectively. 1 mL of the good solvent was added, completely dissolved and then filtered. 0.2 mL of filtrate was taken, the anti-solvent was added dropwise thereto respectively (stop adding if there is a precipitate, and adding 1.8 mL of anti-solvent at most), the mixture was stirred for a period of time, and the filtrate was removed by quick centrifugation to obtain the salt of the compound.
• sulfuric acid, hydroxyethyl sulfonic acid and 1,5-naphthalene disulfonic acid can form salt with the free base of the compound.
• the number of the hydroxyethyl sulfonic acid in the hydroxyethyl sulfonate of the compound of embodiment 13-1 is calculated to be 1.
• the good solvent was selected, the acid was weighed, the good solvent was added thereto to prepare the stock solution containing the compound in the concentration of 100 mg/mL.
• the anti-solvent was added thereto, 100 mg of compound was weighed respectively, 1 mL of the good solvent was added, completely dissolved and then filtered. 0.2 mL of filtrate was taken, the anti-solvent was added dropwise thereto respectively (stop adding if there is a precipitate, and adding 1.8 mL of anti-solvent at most). The mixture was stirred for a period of time, the filtrate was removed by quick centrifugation, the XRPD of a solid was measured after dying.
• the resulting salt forms with crystal forms were hydroxyethyl sulfonate, sulfate, and 1,5-naphthalenedisulfonate.
• the compound of embodiment 13-1 (100 g), isopropanol (1200 mL) were added to a 3 L three-necked flask, heated to 40 to 45°C, stirred to dissolved clarification; and the 2- hydroxyethyl sulfonic acid (28.84 g) was dispersed in 800 mL of ethanol, the ethanol solution was added dropwise to the reaction system at a controlled temperature of 39 to 42°C for about 10 minutes. 500 mg of seed crystal was added to the above reaction mixture and a solid was precipitated rapidly. The heating was removed, the reaction mixture was cooled to 25°C and stirred for 12 hours.
• the reaction mixture was filtered, and the filter cake was washed with 400 mL of isopropanol, drained to dryness and dried under vacuum at 45°C for 16 hours to obtain 92.57 g of a pale yellow solid with a purity of 97.9%, a chiral purity of 92%, and a mass yield of 92%.
• the pale yellow solid has an XRPD pattern as shown in FIG. 1 , a DSC pattern as shown in FIG. 2 , and a TGA pattern as shown in FIG. 3 by detection and analysis.
• Crystal form I 1 Light ( ⁇ 1.2 ⁇ 10 6 lux-h, 10 days) Crystal form I 2 High humidity (25°C, 75%, 10 days) Crystal form I 3 High humidity (25°C, 90%, 10 days) Crystal form I 4 High temperature (40°C, 30 days) Crystal form I 5 High temperature (60°C, 30 days) Crystal form I 6 Micropowder Crystal form I
• crystal form I of hydroxyethyl sulfonate of the compound of embodiment 13-1 was weighed and placed under light (5000 ⁇ 500 lux), high temperature (60°C), high humidity (92.5% RH), and high temperature and high humidity (50°C &75% RH) conditions for 10 days, respectively, and a solution containing free base of the embodiment 13-1 at a concentration of 0.25 mg/mL was prepared by adding diluent methanol, analysed by HPLC, and the change of related substances was calculated according to the peak area normalization method.
• the crystal form II of sulfate is relatively stable under light, high humidity, high temperature and high humidity conditions.
• thermodynamic solubility of the compound at 37°C was measured by HPLC with external standard method.
• thermodynamically stable crystal form of hydroxyethyl sulfonate by screening of polycrystal forms.
• thermodynamically stable crystal form of sulfate by screening of polycrystal forms.

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